Malignant Mesothelioma_

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    [CANCER      57.466-471. February1, 1997]
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                                        Herpes Virus to Treat Experimental
    Use of a “Replication-Restricted―                                                                                                                                                       Human
    Malignant Mesothelioma
    John C. Kucharczuk,' Bruce Randazzo,' Michael Y. Chang, Kunjiata M. Amin, Ashraf A. Elshami,
    Daniel H. Sterman, Nabil P. Rizk, Katherine L. Molnar-Kimber, S. Moira Brown, Alasdair R. MacLean,
    Leslie A. Litzky, Nigel W. Fraser, Steven M. Albelda, and Larry R. Kaiser@
    Thoracic Oncology Research Laboratory Ii. C. K.. M. Y. C., K. M. A., A. A. E., D. H. S.. N. P. R.. K. L M-K.. L A. L. S. M. A., L R. K.] and Department of Dermatology [B. R.].
    University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104; Wistar Institute, Philadelphia, Pennsylvania 19104 (B. R., N. W. F., S. M. A.]: and Glasgow
    University, Neurovirology Research Laboratories, Glasgow, Scotland 651 4TF (S. M. B., A. R. M.]

    ABSTRACT                                                                                                                         (12). This mutation, through mechanisms that are still unclear, se
                                                                                                                                     verely attenuates the ability of HSV-l716 to replicate in normal
       MOdified, nonneurovirulent                        herpes simplex viruses (HSVs) have shown
                                                                                                                                     tissues (12, 17, 18), but does not appear to affect the virus' ability to
    promise in the treatment of brain tumors. However, HSV-1 can infect and
                                                                                                                                     replicate in rapidly dividing malignant cells. We and others (18, 19)
    lyse a wide range of cell types. In this report, we show that HSV-1716, a
    mutant      lacking both copies of the gene coding ICP-34.5,                                         can effectively
                                                                                                                                     have shown that HSV-1716 is extremely nonneurovirulent in animal
    treat a localized Lp malignancy. Human malignant mesothelloma cells                                                              models and can be used to treat intracranial tumors in normal and
    supported the growth of HSV-1716 and were efficiently lysed in vitro. i.p.                                                       immunodeficient mouse models (15, 16, 20).
    injection      of HSV-1716             into animals              with established               tumor     nodules         re        Neurovirulent, wild-type HSV-l is able to infect and lyse a wide
    duced tumor burden and significantly prolonged survival in an animal                                                             variety of cell types. Therefore, we reasoned that a mutant HSV virus,
    model ofnon-central                 nervous system-localized                     human malignancy                  without       such as HSV-17l6, might be efficacious in the treatment of localized,
    dissemination or persistence after i.p. injection Into SC@ mice bearing                                                          non-CNS malignancies. In addition, the use of this virus in a location
    human       tumors.         These findings suggest that this virus may be efficacious                                            distant from the CNS might offer significant safety advantages. Ac
    and safe for use in localized human malignancies of noaneuronal origin                                                           cordingly, in this paper we report on the successful in vitro and in vivo
    such as malignant mesothelioma.
                                                                                                                                     use of HSV-l716 to treat a localized, non-CNS malignancy. We have
                                                                                                                                     chosen human malignant mesothelioma as a model tumor system for
    INTRODUCTION                                                                                                                     HSV-l716 because of the localized nature of this malignancy (allow
                                                                                                                                     ing for direct virus administration) and because of the current lack of
       The recent emergence of viral-based “gene   therapy―as an approach                                                        effective treatment (21).
    to treat cancer has generated a great deal of enthusiasm and interest
    (1). For example, our group and others have explored the use of
                                                                                                                                     MATERIALS             AND METHODS
    retrovirus-producing cells or adenoviruses to deliver the HSV& gene
    into localized malignancies, such as brain tumors and malignant                                                                    HSV-1716. HSV-l716 was originally isolated in the laboratory of S. M.
    mesothelioma (2—6).Although a “bystander―      effect amplifies the                                                        Brown(Glasgow,Scotland)andpassagedfor use in this studyby N. W. Fraser
    efficacy of the HSVtk/ganciclovir system (7), it is likely that a major                                                          (Philadelphia, PA). The genome of this virus contains a 759-bp deletion
    obstacle in treating actual patients with localized malignancies will be                                                         located within each copy of the BamHI fragment of the long repeat region of
    the ability to transduce a sufficient number of cells within a large                                                             the genome (12). These deletions remove most of the gene encoding ICP 34.5,
    tumor mass. One strategy that holds promise is the use of replicating                                                            and the mutant fails to make the protein (12, 22)
                                                                                                                                        In Vitro Studies of HSV-1716 on a Human Malignant Mesothelioma
    viral vectors (8).
                                                                                                                                     Cell Line. A humanmalignantmesotheliomacell line call REN was isolated,
       The idea of using replicating viruses to treat tumors was suggested                                                           characterized, and passaged as described previously by our laboratory (23).
    as early as 1904 by George Dock (9); in fact, a number of clinical                                                               Human lung cancer cell lines A549, H322, and H358 were obtained from the
    trials were conducted                  in the l950s              and 1960s with some success                            (10,     American Type Culture Collection (Rockville, MD). The human osteosarcoma
    11), although interest in the use of viral-based cancer therapy waned                                                            cell line SAOS was obtained from T. Halazonetis (The Wistar Institute). The
    thereafter. Advances in virology and molecular biology now allow the                                                             human melanoma cell line WM451 Lu was obtained from M. Herlyn (The
    “engineering―of viruses                 with specific               properties,          suggesting            that the      Wistar Institute) and was isolated, characterized, and passaged as described
    idea of viral-based cancer therapy should be revisited. One promising                                                            previously (24). To construct single-step viral growth curves, cells were plated
    virus in this regard is HSV-l.                               A number             of HSV-l            mutants          have      on six-well plates at a density of 5 X iO@cells/well and infected 24 h later with
                                                                                                                                     HSV-l7l6 at a MO! of 0.01 (5 X l0@PFU/well). One well was harvested at
    recently been identified that appear to replicate preferentially in
                                                                                                                                     0, 6, 12, and 24 h by cell scraping and collection of the media. The samples
    rapidly dividing transformed cells (12, 13). Because of the natural
                                                                                                                                     were freeze/thawed and titered by plaque assay on baby hamster kidney cell
    tropism of wild-type herpes virus for neuronal tissue, the published                                                             monolayers. A cell viability assay was performed by plating cells in 96-well
    uses of modified,               replicating            HSV to treat cancer have centered                                  on     plates at a density of 5 X l0@ cells per well. Twenty-four h later, the cells were
    tumors of CNS origin (14—16).One of these mutants is HSV-1716, a                                                               infected with HSV-l716 at MOIs of 0, 0.001, 0.01, and 0.1. Six wells were
    virus that contains a 759-bp deletion in the genes coding for ICP 34.5                                                           infected at each MO!. A sufficient number of plates were used to allow for
                                                                                                                                     viability assay at 24, 48, 72, and 96 h after infection. Viable cell number was
       Received 7/29/96; accepted 12/4/96.                                                                                               assessed by a colorimetric assay (CellTiter 96 Aqueous Nonradioactive         MTT
       The costs of publication of this article were defrayed in part by the payment of page                                         Cell Proliferation Assay; Promega, Madison WI) that measures viable cell
    charges. This article must therefore be hereby marked advertisement in accordance with                                               dehydrogenase   activity   by absorbance.   The percentage   of control   growth   is
    18 U.S.C. Section 1734 solely to indicate this fact.
                                                                                                                                         defined as the ratio of the mean absorbance of six treatment wells at 490 nm
       I The    first   two   authors    contributed       equally       to this   work.
       2 To whom        requests    for reprints       should     be addressed,       at Division     of Thoracic       Surgery,         to the mean absorbance of six untreated matched controls.
    University of Pennsylvania Medical Center, 4 Silverstein, 3400 Spruce Street, Philadel                                                  In Vivo Studies. A previously described model of human malignant me
    phia, PA 19104. Phone: (215) 662-7538.                                                                                               sothelioma growing in the peritoneal cavity of SCID mice was used for all in
       3 The    abbreviations      used are: HSVtk,             herpes   simplex     thymidine      kinase;   HSV-l,      herpes
                                                                                                                                         vivoexperimentation (23). Animal protocols were approved by the Animal Use
    simplex-l virus; CNS, central nervous system; ICP, infected cell protein; MO!, multi
    plicity of infection; PFU, plaque-forming unit; MT1', 3-(4,5-dimethylthiazol-2-yl)-2,5                                               committees of the Wistar Institute and the University of Pennsylvania in
    diphenyltetrazolium          bromide.                                                                                                compliance with the Guide for the Care and Use of Laboratory Animals (NIH
                                                                               OF        MESOTHEUOMA
                                                                       TREATMENT MALIGNANT         WITh HS@

    No. 85-23,      revised   1985).   Briefly,   SCID mice were obtained        and housed    at
    the animal facilities of the Wistar Institute. On day 0, animals were injected
    i.p. with 3 X l0@ REN cells in 1 cc of cell culture media. For the tumor
    burden study, treatment animals were given 5 X 106PFU of HSV-1716 in
    culture media by i.p. injection on day 14. Control animals received an
    equivalent volume of culture media. The animals were examined daily and
    sacrificed     by cervical dislocation        on day 28. The amount of tumor burden                             ioe
    was assessed using a four-point semiquantitative scale, which accounts for
    both gross and microscopic disease. Briefly, animals were assessed for
    tumor in the following four areas: stomach/pancreas, portal region, retro
    peritoneum/diaphragm, and small bowel mesentery. On gross examination,
    animals received either a score of 0 if no tumor was present or a score of
    1 in each of the four designated areas where gross tumor was seen. If no
    gross tumor was visible, H&E-stained paraffin-embedded sections of each                                 @o      ::
    organ from the designated area were examined in a blinded fashion by an
    anatomic pathologist. The sections were scored as either 0 for no micro
@   scopic tumor or 0.5 if microscopic tumor was present. Thus, the tumor                                           io2
    scores ranged from 0 to 4.0. Organs including brain, heart, lungs, liver,
    stomach, pancreas, kidney, adrenals, spleen, gonads, small bowel,                         and                   10
    diaphragm were obtained from each animal. Each organ was divided into
    thirds with equal samples designated                for frozen section, formalin fixation,                      100
    and DNA extraction.                                                                                                              0            6             12                 24
       For the initial survival study, animals were injected i.p. with 3 X l0@REN                                                                            (hours)
                                                                                                                                             Timepost intec@on
    cells in 1 ml of cell culture media (day 0). On day 7, one animal was sacrificed
    for gross tumor confirmation, and the remaining animals were randomized to                          B
    the treatment group and the control group. Treatment animals received 5 X 106                                    100
    PFU of HSV-l7l6 by i.p. injection; control animals received an equal volume
    of culture media. The animals were checked daily and followed for survival.
    The same protocol was followed for the dose-response study, except the
    animals      were randomized       into a control     group,   a high dose group   (5 X 106                          80

    PR! HSV-l716), a middle dose group(5 X l0@'    PFU HSV-l7l6), and a low
    dose group (5 X 10@   PFU HSV-l7l6). A similar protocol was followed to                                                                                                  -K— M.O.I. = 0.0
    evaluate the early deaths observed in the survival studies, except that the                                                                                              -.-   M.O.I. = 0.01
                                                                                                                         60                                                  -A-   M.O.$.   = 0.1
    tumor-bearing      animals were sacrificed 3 days after viral administration.        Blood
                                                                                                                                                                             -.-   M.O.I. -1
    samples were obtained, pooled, and evaluated for serum creatinine and serum                             .0
    uric acid level. Serum chemistry analysis was performed in the Pepper Clinical                          >
    Laboratory at the University of Pennsylvania Medical Center (Philadelphia,                                           40

       Histology and Immunohistochemistry. As reported previously, the REN
    cell line was isolated from a patient with the appropriate clinical and patho
    logical features of malignant mesothelioma, including morphological and                                              20

    immunohistochemical staining pattern (CEA, LeuMl, and mucicarmine anti
    body negative; Ref. 23). To further assure the nonneuronal origin of these cells
    for the purposes of this study, REN-derived tumors were stained immunohis
    tochemically with an antibody called TA-5l (a gift of J. Q. Trojanowski,                                                                    24            48             72                     96

    University of Pennsylvania, Philadelphia, PA), which recognizes neurofila                                                                                  (Hours)
    ment H, a specific marker for neuronal lineage (25). REN cells were TA-S1
    negative (data not shown). Immunohistochemical staining for HSV antigens                              Fig. 1.A, HSV-17l6 single-step viral growth curve on human malignant mesotheioma
                                                                                                       cells. Inoculum at time 0 was 5000 PR] of virus (MOl = 0.01). Note that at 24 h the
    was performed on paraffin sections of tissues fixed in 10% phosphate-buffered                      amount of virus present had increased by fourlogs over the initial input inoculum. B, MU
    formalin, using a commercially available rabbit anti-HSV polyclonal antibody                       assay for human malignant mesotheioma cell viability as a function of time and varying
    (DAKO, Carpinteria, A).
                          C                                                                                            o                i                                     cells
                                                                                                       MOl.Thepercentage fcontrolsurvival stheratioofmeanMTI'activityininfected
                                                                                                       (n       6 wells at each time point) to the mean activity in matched uninfected cells (n = 6
       In Vivo Dissemination and Restriction Studles To look for dissemina                             wells at each time point).
    tion of HSV-l7l6, we performed PCR looking for the HSVtk gene in the
    collected tissues. Genomic DNA was obtained by standard phenol/chloro
    form extraction       and amplified       by PCR. The PCR primers            (sense ATG
    GCTITFCGTACCCCTGCCAT and antisense GGTATCGCGC0000G-                                                RESULTS
    GGTA) were designed to span a region of the HSVtk gene generating a
                                                                                                          HSV-1716 Efficiently Replicates in a Human Malignant Me
    536-bp fragment. The genomic DNA extracted from each tissue sample was
    subjected to 35 cycles of PCR using the tk primers. The tk plasmid and                             sothelioma Cell Line and Lyses the Cells in Vitro. To determine the
    DNA extracted from brain tissues of an animals infected with wild-type                             ability of HSV-1716 to replicate within a non-CNS human tumor cell
    HSV- 17+ were used as positive controls for the PCR reaction. PCR                                  line in vitro, we performed a single-step viral growth curve in REN
    products were run on ethidium bromide containing 1.5% agarose gels, and                            cells. As shown in Fig. 1A, REN cells supported efficient growth of
    the positives produced the expected 536-bp fragment. The gels were then                            1716. Immediately after infection (time 0), 70% of the input viral
    overnight blotted onto Zeta-Probe GT blotting membranes (Bio-Rad Lab                               inoculum was recovered. By 6 h, the number of recovered active viral
    oratories, Hercules, CA). The membrane was UV cross-linked and probed                              particles fell by a factor of 200, as expected due to viral uptake and
    using a 32P-Iabeled portion of the HSVtk plasmid.                                                  disassembly in preparation for viral replication. However, at 24 h, a
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                                                                                                                                                  OF        MESOTHELIOMA@1TH
                                                                                                                                          TREATMENT MALIGNANT              HSV

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        Fig. 2. Immunohistochemistry for HSV viral proteins in SC@ mice. Micrographs 3—5 after i.p. injection of wild-type virus HSV-17+ . Positive immunostaining (brown cells)
    is seen in the normal tissues, including ganglia of the myenteric plexus (a), adrenal gland (b), and brain (c). In contrast, d shows a tumor-bearing animal 72 h after receiving 5 X l0@
    PFU of HSV-17l6 by i.p. injection. Note the marked positivity of tumor cells with the lack of infection in the surrounding normal tissues (X200, arrowheads mark the interface between
    tumor (1) and normal tissues).

    4-log increase over the initial inoculum was observed demonstrating                                                                                                                                       mcd, the human lung cancer lines A549 and H322, were lysed poorly
    highly efficient replication of HSV-1716 on REN cells.                                                                                                                                                    by both HSV-l716 and wild-type HSV-l7+ (data not shown).
       To demonstrate the ability of HSV-17l6 to directly kill REN cells,                                                                                                                                        In Contrast to Wild-Type HSV-17+, HSV-1716 Infection and
    we performed an in vitro cell viability assay at various times after                                                                                                                                      Replication Is Restricted to Tumor Cells in an in Vivo SC@
    viral infection. As shown in Fig. lB. HSV-1716 efficiently killed                                                                                                                                         Mouse Model of Human Mesothelioma. As expected, i.p. injection
    target REN cells in a time- and dose-responsive fashion. At 96 h                                                                                                                                          of SCID mice with 5 X 106 PFU of wild-type HSV-17+ led to rapid
    postinfection, there were no viable tumor cells remaining in cultures                                                                                                                                     spread of the virus, signs of encephalitis (i.e., ataxia and hind limb
    infected at MOIs ranging from 0.01 to 1.0. Similar results were                                                                                                                                           paralysis),             and death of all animals by 7 days. To determine                                                                                      the extent
    obtained for several other nonneuronal cell lines tested in this viability                                                                                                                                of HSV infection, organs from animals sacrificed 3—5     days after
    assay, such as the human osteosarcoma line SAOS, the human lung                                                                                                                                           wild-type HSV-HSV-17+        injection were analyzed immunohisto
    cancer line H358, and the human melanoma cell line WM 45 1 Lu                                                                                                                                             chemically with a polyclonal antibody recognizing HSV antigens.
    (data not shown). However, two other nonneuronal cell lines exam                                                                                                                                          Reactive cells were clearly seen in the myenteric ganglia of the small
                                                           OF        MESOTHELIOMA ITHHSV
                                                   TREATMENT MALIGNANT          W

intestine (Fig. 2A), adrenal glands (Fig. 2B), and brain (Fig. 2C). In          period. There was one death in the treatment group that occurred 5
contrast to wild-type HSV-HSV-l7@, SCID mice (n = 5) injected                   days after viral administration from unknown cause.
with 5 X 106 PR] of HSV-l716 remained alive at 100 days without                    To determine whether the decrease in tumor mass conferred a
signs of encephalitis. Immunohistochemistry for HSV antigens was                survival advantage to SCID mice bearing established i.p. REN tumors,
negative at days 3, 8, and 14 in all tissues analyzed. Specifically, liver,     a group of tumor-bearing animals were injected with 5 X 106 pp@j of
kidney, adrenal, spleen, small bowel myenteric plexuses, and brain              HSV-17l6 2 weeks after i.p. injection of tumor cells. The animals
were negative for HSV antigen.                                                  were followed for survival. The median survival was increased from
   To assess the ability of HSV-17l6 to infect and replicate within             47 days in the control group (n = 9) to 95 days in the treatment group
human tumors in vivo, SCID mice were injected i.p. with 30 million              (n     10). All deaths in the control group were a result of bulky i.p.
human REN cells. After 14 days, diffuse macroscopic 5—8-mm         tumor      disease;   no external     tumor   nodules   were visible   at the initial   tumor
nodules were present. At this time, 5 X 106 PFU of HSV-17l6 were                injection site. It is interesting that deaths in the treatment group
instilled into the peritoneal cavity, and the animals were sacrificed                                                                          d
                                                                                occurred at two distinct time points. Three animals died 2—5ays after
72 h later. Microscopic examination revealed that virtually all tumor           HSV-1716 administration. There was no evidence of bulky disease at
                                                                                this time. The majority of the remaining treated animals died around
nodules showed necrosis, multinucleated cells, and nuclear inclusions
                                                                                day 100 due to bulky malignant disease that extended from large s.c.
consistent with active herpetic infection. Infiltration with mononu
                                                                                nodules arising on the anterior abdominal wall. At 102 days, the three
clear inflammatory cells was also present. In contrast, no viral cyto
                                                                                remaining treatment animals were sacrificed and necropsied. These
pathic changes were seen in normal tissues. To directly detect HSV
                                                                                animals also had nodules corresponding to the site of the initial tumor
infection, tumor tissue and organs were stained with an anti-HSV
                                                                                injection, and tumor appeared to be growing inward from the anterior
polyclonal antibody. Fig. 2D shows a representative tumor nodule
                                                                                abdominal injection site with invasion into the peritoneal cavity.
growing on the inferior aspect of a SCID mouse stomach. Three days
                                                                                   A second survival study was performed to determine the viral dose
after HSV-l716 administration, a large percentage of the tumor cells
                                                                                response. Tumor-bearing        animals were randomized to control
stained positively for HSV antigens, whereas surrounding normal                 (n     11) and treatment groups (low dose: 5 X l0@ PHi HSV-l716,
tissues, as well as all other normal tissues examined, showed no                n     10; middle dose: 5 X l0@ PHi HSV-l7l6, n            10; high dose:
positive staining. Specifically, liver, kidney, adrenal, spleen, small          5 x 106 PHi HSV-l716, n          10). As shown in Fig. 4, treatment with
bowel myenteric plexuses, and brain were negative for HSV antigen.              high dose HSV-17l6 significantly improved survival when compared
Similar results were obtained at days 5, 7, and 14 after infection;             with control     animals    (P = 0.001 1 by Mantel-Cox            log-rank    test).
however, the number of virally infected tumor cells appeared to                 Animals in the high dose group had a mean survival of 102 days;
decrease at the later time points, possibly due to a decrease in                again, surviving animals developed s.c. tumor nodules on the anterior
available tumor substrate.                                                      abdominal wall corresponding to the initial tumor injection site by day
   To more sensitively monitor the dissemination of HSV-1716 in                 100. Six of these animals subsequently died from bulky tumor ex
tumor-bearing animals, we used the PCR followed by Southern blot                tending from the anterior abdominal wall injection site into the peri
ting to detect the HSVtk gene. This assay can detect HSV DNA at a               toneal cavity. The low and middle dose treatment animals also dem
level of sensitivity equivalent to one genome copy per 100,000 cells.4          onstrated a significant improvement in survival when compared with
Fig. 3A shows the results from a tumor-bearing mouse 3 days after i.p.          the control animals (P = 0.0003 for control versus middle dose and
installation of 5 X 106 PFU of HSV-l 716. HSV DNA was detected in               P = 0.0019 for control versus low dose by Mantel-Cox log-rank test).
the tumor nodule (Fig. 3A, Lane 6) at this time; however, no viral              There was no difference in survival between the low and middle dose
DNA was detected in brain, spinal cord, adrenal gland, lung, or liver,          animals (P = 0.65).
confirming the immunohistochemistry results. In contrast, wild-type                In both of the survival studies, approximately 20% of tumor
HSV led to detectable HSVtk sequences in the brain (Fig. 3B, Lane 1).           bearing    animals   died suddenly    2—5days after HSV-l716       administra
Fig. 3B shows the results from two tumor-bearing animals 14 days                tion. Because of autolysis, only limited autopsies could be performed,
after i.p. injection of HSV-17l6. Again, no viral DNA was detected in           and the cause of these early deaths remains unknown. These animals
any of the normal tissues assayed.                                              did not display signs of encephalitis (i.e., no ruffled fur, no paralysis,
   HSV-1716 Reduces i.p. Tumor Burden and Increases Survival                    and no ataxia) before their deaths. Immunohistochemistry and PCR
in a SC@ Mouse Model of Human Mesothelioma. To determine                        data (see above) ruled out widespread viral dissemination. To study
                                                                                the possibility that early deaths in treated tumor-bearing animals were
the ability of HSV-l7l6 infection to eradicate established tumor,
                                                                                due to acute renal failure after massive tumor lysis, serum creatinine
animals that had previously received 30 million i.p. REN tumor cells
                                                                                and uric acid were also studied. The serum creatinine level in a pooled
were given 5 X 106 PFU of HSV-1716 by i.p. injection. At the time
                                                                                specimen from four tumor-bearing animals 3 days after HSV-l716
of viral administration, animals had established i.p. tumors consisting
                                                                                administration was 0.2 mg/dl, and the uric acid level was 4.0 mg/dl.
of multiple 5—8-mmnodules with portal invasion and gall bladder
                                                                                The serum creatinine level in matched untreated tumor-bearing mice
distention. Two weeks later, animals were sacrificed, and the tumor
                                                                                was 0.3 mg/dl, and the uric acid level was 3.9 mg/dl.
burden was assessed using a previously developed semiquantitative
scale that accounts for both gross and microscopic tumor (23). The
tumor score ranges from 0 (no gross or microscopic tumor) to a                  DISCUSSION
maximum score of 4.0. There was a significant reduction in the mean                In this report, we demonstrate that the mutant “replication-restrict
tumor score at day 28 in tumor-bearing animals (n = 12) treated with                  H
                                                                                ed― SV-1716 (an ICP 34.5 null mutant) can reduce tumor burden
HSV-l716 compared with the mean tumor score in control animals                  and significantly prolong survival in an animal model of localized
(n     8). The mean tumor score in the treatment group was 1.4 ±0.2            non-CNS human malignancy. Furthermore, we show that after i.p.
compared with a mean tumor score in the control group of 3.9 ±0.1              injection of HSV-1716 into SCID mice bearing human tumors, rep
(P < 0.001). All animals in the control group survived the study                lication is restricted to the tumor and does not disseminate or replicate
                                                                                outside the tumor. Although HSV is normally a neurotropic virus,
  4 K.   Amin,   unpublished   data.                                            these findings highlight the tumor-killing potential of HSV-l716 for
                                                                    TREATMENT MAUGNANT           W
                                                                                      ME5OTHELIOMA ITHHSV

                                                                                               unknown but are under active investigation in a number of laborato
    A                                                                                          ries. The JCP 34.5 gene encodes a protein of 263 amino acids
                                                                                               consisting of a large amino-terminal domain, a linker region of three
@                          1.         tkplesmld           control                              amino acid repeats (Ala-Thr-Pro, AlP codons), and a carboxyl
                          2.         breln                                                     terminal domain (27). The carboxyl-terminal domain is homologous
                                                                                               to domains of MyD1 16 and growth arrest and DNA damage gene 34
                          3.         spInalcord                                                (GADD 34; Refs. 28 and 29), cellular genes involved in cell cycle
                          4.         lIver                                                     regulation and programmed cell death. Recent work suggests that the
                                                                                               HSV ICP 34.5 gene precludes cells from triggering total protein
                          5.         adrenal                                                   synthesis characteristic of programmed cell death, at least in a neu
                                                                                               roblastoma system (30). However, this does not appear to be a
         ,               - 6.        tumor                                                     universal response, and at this time all that can be said is, that in
@                                    lung                                                      contrast to normal cells, malignant cells have abnormalities in cell
                                                                                               cycle and/or protein synthesis pathways that appear to render them
                          6.         DRG                                                       permissive for viruses lacking the ICP 34.5 gene. We have recently
                          9.           watercontrol
                                     PCR                                                       demonstrated in a range of primary human glioblastoma cells, that
                                                                                               permissivity for HSV-1716 replication correlates positively with the
                                                                                               rapidly dividing nature of the cells (26).
             S              1. 17+breln                                                           The ultimate clinical utility of replication-restricted viruses such as
                                                                                               HSV-1716 will be determined by the balance between efficacy and
                     .     2.        PCR      water       control
                                                                                               safety. The experiments presented here, and by others in brain tumor
                                                                                               models, indicate that mutant herpes viruses can selectively replicate in
                           3.        BraIn                                                     human and murine malignant cells to induce the regression of estab
                                                                                               lished tumors. Two factors inherent in an HSV-based approach to
                           4.        lungs                                                     mesothelioma therapy provide potentially valuable safety features. (a)
@                          5.        heart                              Animal 1               The retention of the HSVtk gene in HSV-l716 will allow treatment
                                                                                               with the antiviral drugs acyclovir or ganciclovir at any point to abort
               ‘         6.        lIver                                                     the infection. (b) Administration of virus to a peripheral location, such
                                                                                               as the pleural or peritoneal cavity rather than directly into the brain,
                           7.        ovary                                                     should increase the margin of safety.
                           8.        braIn                                                        One issue that will likely affect the efficacy of replication-restricted
                                                                                               HSV mutants will be the immune response of the host to virus. A
                                                                                               critical balance between optimal viral replication leading to tumor
                           9.        heart                              Animal 2               destruction and the immune-mediated elimination of viral infection
                            10. lungs                                                          must be achieved. The efficacy of replication-restricted HSV infection
                            11.      lIver                                                     in immunocompetent animals with non-CNS malignancy, and even
                                                                                               more importantly in immunocompetent HSV-primed animals, will
                 @&         12.      ovary
                                                                                               need to be analyzed.
                                                                                                  Safety issues, specifically the possibility of viral dissemination
         •iJV.@--@ tkPlesmld

       Fig. 3. A, PCR using primers for the HSVtk gene were performed on a control plasmid
    containing the HSVtk gene (lane 1) and from DNA extracted from tissues in a tumor
    bearing animal 3 days after i.p. HSV-1716 virus administration(Lanes2—8). he Southern
    blot demonstrates HSVtk signal in the tumor nodule and lack of signal in normal tissues.
    DRG, dorsal root ganglion. B, PCR using primersfor the HSVtk gene were performed              .8
    from DNA extracted from the brain of an animal 5 days after i.p. injection of wild-type
    HSV-l7 (Lane 1), from tissues of tumor-bearing    animals 14 days afteri.p. virus admin                                                                x Contxol
                                                                                                       Viral Treatment
                        and                             the
    istration(Lanes3—12), on a controlplasmidcontaining HSVtkgene(Lane13).                                                                               U HighDoseVirus
    The Southern blot demonstrates the presence of viral HSVtk in the brain of the animal      I .6                                                        A

                                                                                                                                                               Mi@ Dose   Virus

    infected with wild-type virus. In contrast, there is no HSV DNA detected in tissues from
    animals treated with HSV-l7I6.

                                                                                               (@ .4

    localized malignancies of nonneuronal origin such as malignant me
    sotheioma. In addition, we found that a variety of nonneuronal tumor
    cell types are susceptible to HSV lysis in vitro, but that susceptibility
    is not universal. Recently, we have shown that some cell lines derived
    from tumors of the CNS can also be essentially resistant to lyric                 0
    infection by wild-type HSV and/or ICP 34.5 null mutants (26). We are                   0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
    presently screening a large number of tumors of both neuronal and
                                                                                                           Time post tumorcell injection(days)
    nonneuronal lineage with the goal of better defining the cellular
    factors that restrict lyric HSV infection.                                       Fig. 4. HSV-l716 viral dose-response survival study. SC@ mice received 3 X l0@
       The “replication-restriction― marked attenuation of neuroviru
                                     and                                          human malignant mesothelioma cells on day 0. Seven days later, one animal was
                                                                                  sacrificed to confirm tumor. The remaining animals were randomized into four groups:
    lence of HSV-1716 results from deletion of the gene coding for ICP control(n = ll,culturemedia),lowdose(n = 10,5 X lO@PFUHSV-17l6),middledose
    34.5. The precise mechanisms responsible for these properties are still       (n     10, 5 X 10' PFU HSV-1716), and high dose (n = 10, 5 X 1O@  PFU-l7l6).
                                                                      OF        MESOTHELIOMA
                                                              TREATMENT MALIGNANT          WITHHSV

resulting in CNS infection with or without neural latency, will also                                  on the use of viruses in the treatment of carcinoma of the cervix. Cancer (Phila.), 9:
                                                                                                      1211—1218, 1956.
determine the ultimate clinical utility of replication-restricted viruses
                                                                                                  12. MacLean, A. R., Ul-Fareed, M., Robertson, L., Harland, J., and Brown, S. M. Herpes
such as HSV-1716. Administration of HSV-1716 to the peritoneal                                        simplex virus type 1 deletion variants 1714 and 1716 pinpoint neurovirulence-related
cavity did not result in detectable viral dissemination as evaluated by                               sequences in Glasgow strain 17+ between immediate early gene 1 and the “a―
                                                                                                      sequence. J. Gen. Virol, 72: 631—639, 1991.
immunohistochemistry or a sensitive PCR assay. Specifically, no viral                             13. Chou, J., Kern, E. R., Whitley, R. J., and Roizman, B. Mapping of herpes simplex
proteins or DNA were identified in the spinal cord or brain at 3 or 14                                virus neurovirulence to gamma 1 34.5, a gene nonessential for growth in culture.
days after treatment. Our inability to detect the presence of HSV-17l6                                Science (Washington DC), 250: 1262—1266,1990.
                                                                                                  14. Martuza, R. L., Malick, A., Markert, J. M., Ruffner, K. I., and Coen, D. M.
in normal tissues of treated mice suggests that the replication of HSV                                Experimental therapy of human glioma by means of a genetically engineered virus
JcP 34.5 gene mutants outside the tumor is severely restricted. In                                    mutant. Science (Washington DC), 252: 854—856, 991.1
work reported elsewhere, we have transplanted human skin onto SCID                                15. Markert, J. M., Malick, A., Coen, D. M., and Martuza, R. L. Reduction and elimi
                                                                                                      nation ofencephalitis in an experimental glioma therapy model with attenuated herpes
mice and infected the grafts with HSV-17 16, wild-type HSV- 17+, or                                   simplex mutants that retain susceptibility to acyclovir. Neurosurgery (Baltimore), 32:
a revertant (HSV-1716R in which the ICP 34.5 deletion has been                                                 1993.
repaired; Ref. 31). Wild-type HSV-17+ and revertant virus (HSV                                    16. Randazzo, B. P., Kesari, S., Gesser, R. M., Alsop, D., Ford, J. C., Brown, S. M.,
                                                                                                      MacLean, A. R., and Fraser, N. W. Treatment of experimental intracranial murine
1716R) replicated briskly within the human skin and led to ulceration                                 melanoma with a neuro-attenuated herpes simplex virus-l mutant. Virology, 211:
and gross destruction of the grafts. In contrast, replication of HSV                                          1
                                                                                                      94—101, 995.
1716 was severely restricted in this normal human tissue homologue.                               17. Robertson, L. M., MacLean, A. R., and Brown, S. M. Peripheral replication and
                                                                                                      latency reactivation kinetics of the non-neurovirulent herpes simplex virus type-l
   Approximately 20% of HSV-l7l6-treated tumor-bearing mice died                                                                                 1992.
                                                                                                      variant 1716. J. Gen. Virol., 73: 967—970,
shortly after viral inoculation. We did not detect dissemination of                               18. Spivack, J. G., Fareed, M. U., Valyi-Nagy, T., Nash, T. C., O'Keefe, J. S., Gesser,
HSV-17l6 by immunohistochemistry or PCR in mice at these time                                         R. M., McKie, E. A., MacLean,A. R., Fraser,N. W., and Brown,S. M. Replication,
                                                                                                      establishment of latent infection, expression of the latency-associated                    transcripts and
points. Although these deaths are likely due to host-tumor-virus                                      explant reactivation of herpes simplex virus type 1 gamma 34.5 mutants in a mouse
interactions, we were unable to establish their exact etiology. Tumor                                 eye model. J. Gen. Virol., 76: 321—332,1995.
lysis appears to be an unlikely cause, because the creatinine and uric                            19. Valyi-Nagy, T., Fareed, M. U., O'Keefe, J. S., Gesser, R. M., MacLean, A. R.,
                                                                                                      Brown, S. M., Spivack, J. G., and Fraser, N. W. The HSV-l strain 17+ gamma 34.5
acid levels in the serum of treated mice did not differ from untreated                                                                                                               1994.
                                                                                                      deletion mutant 1716 is avirulent in SCID mice. J. Gen. Virol., 75: 2059—2063,
tumor-bearing controls. Clearly, a more complete understanding of                                 20. Kesari, S., Randazzo, B. P., Valyi-Nagy, T., Huang, Q. S., Brown, S. M., MacLean,
this phenomenon, as well as other potential side effects, will be                                     A. R., Lee, V. M-Y., Trojanowski,J. Q., and Fraser,N. W. Therapyof experimental
                                                                                                      human brain tumors using a neuroattenuated herpes simplex virus mutant. Lab.
important before the clinical use of this or other HSVs is attempted.                                                        1
                                                                                                      Invest., 73: 636—648, 995.
                                                                                                  21. Rusch, V. W. Pleurectomy/decorticationand adjuvant therapy for malignant mesothe
                                                                                                      lioma. Chest, 103: 382S—384S,  1993.
REFERENCES                                                                                        22. McKie, E. A., Hope, R. G., Brown, S. M., and MacLean, A. R. Characterization of
                                                                                                      the herpes simplex virus type 1 strain 17+ neurovirulence gene RL1 and its expres
 1. Vile, R., and Russell, S. Gene transfer technologies for the gene therapy of cancer.
                                                                                                      sion in a bacterial system. J. Gen. Virol., 75: 733—741,1994.
    Gene Therapy, 1: 88—98,  1994.
                                                                                                  23. Smythe, W. R., Kaiser, L. R., Amin, K. M., Pilewski, J. M., Eck, S. L., Wilson, J. M.,
 2. Takamiya, Y., Short, M. P., Moolten, F. L., Fleet, C., Mineta, R., Breakefield, X. 0.,
                                                                                                      and Albelda, S. M. Successful adenovirus-mediated gene transfer in an in vivo model
    and Martuza,R. L. An experimentalmodel of retrovirusgene therapyfor malignant
    brain tumors. J. Neurosurg., 79: 104—1 1993.                                                    of human malignant mesothelioma. Ann. Thorac. Surg., 57: 1395—1401,       1994.
 3. Culver, K. W., Ram, Z., Wallbridge, S., Ishii, H., Oldfield, E. H., and Blaese, R. M.         24. Juhasz, I., Albelda, S., Elder, D., Murphy, G., Adachi, K., Herlyn, D., Valyi-Nagy, I.,
      In vivo gene transfer with retroviral vector-producer cells for treatment of experi             and Herlyn, M. Growth and invasion of human melanomas in human skin grafted to
   mental brain tumors. Science (Washington DC), 256: 1550—1552,       1992.                                                                             1
                                                                                                      immunodeficient mice. Am. J. Pathol., 143: 528—537, 993.
4. Boviatsis, E. J., Chase, M., Wei, M. X., Tamiya, T., R. K. Hurford, J., Kowall, N. W.,         25. Fung, K. M., and Trojanowski, J. Q. Animal models of meduloblastomas and related
   Tepper, R. I., Breakefield, X. 0., and Chiocca, E. A. Gene transfer into experimental              primitive neuroendocrine tumors. J. Neuropathol. Exp. Neurol, 54: 285—296,1995.
   brain tumors mediated by adenovirus, herpes simplex virus (HSV), and retrovirus                26. McKie, E. A., MacLean, A. R., Lewis, A. D., Cruickshank, G., Rampling, R., Barnett,
   vectors. Hum. Gene Ther., 5: 183—191,   1994.                                                    S. C., Kennedy, P. G. E., and Brown, S. M. Selective in vitro replication of herpes
5. Smythe, W. R., Hwang, H. C., Amin, K., lick, S. L., Wilson, J. M., Kaiser, L. R., and              simplex virus type 1 (HSV-l) 1CP34.5 null mutants in primary human CNS tumors
                                                                                                      - evaluation   of   a potentially   effective   clinical   therapy.   Br.   J.   Cancer,    74:   745—752,
      Albelda, S. M. Successful treatment of experimental human mesothelioma using
      adenovirus transfer of the herpes simplex-thymidine kinase gene. Ann. Surg., 222:       1996.
      78—86, 1995.                                                                      27. Chou, J., and Roizman, B. The yl 34.5 gene of herpes simplex virus 1 precludes
 6.   Smythe, W. R., Hwang, H. C., Amin, K. M., Eck, S. J., Wilson, J. M., Kaiser, L. R.,     neuroblastoma cells from triggering total shutoff of protein synthesis characteristic of
      and Albelda,S. M. Recombinant     adenovirus transferof the HSV-thymidineinase
                                                                                   k          programmed cell death in neuronal cells. Proc. Nail. Acad. Sci. USA, 89: 3266—3270,
      gene to thoracicneoplasms:an effective in vitro drug sensitizationsystem. Cancer        1992.
      Res., 54: 2055—2059, 1994.                                                        28. McGeoch, D. J., and Barnett, B. C. Neurovirulence factor. Nature (Land.), 353: 609,
 7.   Freeman, S. M., Abboud, C. N., Wahrtenby, A., Packman, C. H., Koeplin, D. S.,           1991.
      Moolten, F. L., and Abraham, G. N. The “bystander effect:―
                                                                   tumor regression when  29. Lord, K. A., Hoffman-Liebermann, B., and Liebermann, D. A. Sequence of MyD116
      a fraction of the tumor mass is genetically modified. Cancer Res., 53: 5274—5283,     cDNA: a novel myeloid differentiation primary response gene induced by 1L6.
      1993.                                                                                   Nucleic Acids Res., 18: 2823—2828,   1990.
 8.   Russell, S. Replicating vectors for gene therapy of cancer: risks, limitations, and 30. Chou, J., and Roizman, B. Herpes simplex virus 1 yl 34.5 gene function, which
      prospects. Eur. J. Cancer, 30: 1165—1 1994.                                           blocks the host response to infection, maps in the homologous domain of the genes
9.    Dock, G. Influenceof complicatingdiseases upon leukemia.Am. J. Med. Sci., 127:          expressed during growth arrest and DNA damage. Proc. NatI. Acad. Sci. USA, 91:
      536—592, 1904.                                                                                           1994.
10. Asada, T. Treatmentof human cancer with mumps virus. Cancer (Phila.), 34:                     31. Randazzo, B., Kucharczuk, J. C., Litzky, L., Kaiser, L., Brown, S., MacLean, A.,
    1907—1928,                                                                                      Albelda. S., and Fraser, N. Herpes simplex 1716 - an ICP 34.5 mutant - is severely
11. Smith, R. R., Huebner, R. J., Rowe, W. P., Schatten, W. E., and Thomas, L. B. Studies             replication restricted in human skin xenografts in vivo. Virology, 223: 392—396,1996.